PROJECT SUMMARY Cell migration is important for normal biological processes including embryonic development and wound healing, as well as pathological responses such as cancer metastasis. Cyclase-Associated Protein (CAP) is conserved in eukaryotes as a versatile actin-regulating protein. Mammalian CAP1 regulates the actin cytoskeleton and cell migration, and is also implicated in the invasiveness of human cancers. However, roles in cell migration and cancer invasiveness appear to be context-dependent, with insufficiently defined mechanisms. In that vein, our finding of a novel function for CAP1 in cell adhesion provided a mechanism that likely underlies the distinct roles for CAP1 in cell migration and cancer invasiveness. Moreover, we identified the very first regulation mechanism for CAP, where phosphorylation on both residues of the S307/S309 tandem site controls CAP1 association with cofilin and actin. Our latest preliminary studies provide compelling evidence that transient phosphorylation is required for CAP1 to promote cellular actin filament turnover and regulate integrin-mediated cell adhesion. Moreover, the Cyclin-Dependent Kinase 5 (CDK5) and cAMP antagonistically regulate S307/S309 phosphorylation on CAP1. However, critical gaps remain in molecular mechanisms underlying the phosphor-regulation of CAP1 by CDK5 and cAMP, and roles and mechanisms for this regulation in mediating the cell signals to control cell adhesion and migration. We propose this project to fill these critical gaps. Our long-term goal is to understand how cell signaling controls the actin cytoskeleton, cell migration and cancer invasiveness. The overall hypothesis is that CDK5 and cAMP signals antagonistically regulate CAP1 phosphorylation to facilitate spatial-temporally controlled phosphorylation, which is believed to be critical in regulating actin dynamics, cell adhesion and migration. We propose to pursue three specific aims: (1) further determine molecular mechanisms underlying the phosphorylation of CAP1 by CDK5 and dephosphorylation induced by cAMP, respectively; (2) ascertain roles for the phosphor-regulation of CAP1 in mediating CDK5 and cAMP signals to control cell adhesion and migration; (3) determine if S307/S309 phosphorylation regulates CAP1 association with FAK, talin or Rap1, which may underlie the phosphor- regulation of CAP1 function in cell adhesion. The proposed project is significant because successful completion of the specific aims will help us much better understand roles and mechanisms of the phosphor- regulation of the cytoskeletal protein CAP1, which links CAP1 phosphor-regulatory signals to the actin- dependent functions in adhesion and migration. The expected findings will not only greatly extend our knowledge on the regulation of CAP1 cell functions, but may also lead to strategies targeting CAP1 and/or its regulatory cell signals for suppressing the invasive cycle of cancer. This project is highly innovative, because it represents a further departure from the status quo, namely the already paradigm-shifting ongoing studies in our laboratory over the last several years, particularly that on the phosphor-regulation of CAP1.